The invention relates to a process for separating viruses of different sizes, with the virus-containing solution preferably being filtered through one or more filter membranes.
The original aim of gene therapy was to cure genetic diseases by altering body cells genetically in a suitable manner. Nowadays, the term gene therapy is extended to include genetically altered cells which can also be employed therapeutically for curing diseases which do not: have a genetic origin, such as viral diseases, for example.
The genetic alteration of therapeutically active cells requires suitable methods for transferring the nucleic acid, i.e. DNA or RNA, which brings about the genetic alteration of the cell. The nucleic acids which are to be transferred are frequently also described as being so-called transgenes, even when they do not exercise the functions of a gene, e.g. when they are anti-sense nucleic acids. In addition to the direct gene transfer of so-called naked nucleic acids, genetically altered viruses have also proved to be suitable for effecting the gene transfer. At present, retroviruses, adenoviruses or adeno-associated viruses (AAVs), inter alia, are being genetically altered so that they can each be used as a carrier (viral vector) of the transgene(s) for the gene transfer. An important consideration when developing suitable viral vectors is that of the safety aspects when using these vectors in gene therapy. In general, therefore, replication-deficient viruses are developed, that is viruses which, while being able to infect a cell and transfer the transgene(s) into the cell, are unable themselves to replicate in this cell. This is achieved, for example, by deleting the genes which are important for virus replication, for example the genes encoding structural proteins, and, where appropriate, incorporating the transgene(s) in place of them. The preparation of relatively large quantities, which are suitable for the use in gene therapy, of replication-incompetent viruses requires so-called helper viruses, which compensate, in the cell, for the defect in a replication-incompetent virus. The following examples of retroviral vectors and adeno-associated vectors are intended to clarify the general principle:
Replication-deficient retroviral vectors are derived from wild-type retroviruses, which constitute a separate family of eucaryotic viruses whose genetic material (structural genes and regulatory genes) is composed of single-stranded RNA. The viruses are composed of spherical, enveloped virus particles having a diameter of approx. 80-120 nm and an inner capsid, which contains two copies of the genomic RNA in the form of a ribonucleo-protein. For preparing retroviral vectors, one or more structural genes (gag, pol and/or env) is/are replaced by the transgene(s). The LTR (long terminal repeat) regions which are still present at the 5xe2x80x2 and 3xe2x80x2 ends contain, as cis-active elements, regulatory sequences such as a promotor, polyadenylation signals and the sequences which are required for integration into the genome. It is possible, therefore, for the retroviral vector only to contain the LTR regions which flank the transgene(s) The replication of a replication-deficient retroviral vector therefore requires, for example, a helper virus which contains one or more of the abovementioned retroviral structural genes and thus complements the deleted structural gene(s) (Whartenby, K. A. et al. (1995) Pharmac. Ther., 66, 175-190).
Replication-deficient adeno-associated viral vectors are derived from the wild-type AAV, which is a non-autonomously replicating representative of the parvoviruses and constitutes a single-stranded DNA virus having a diameter of approx. 25 nm. Today, it is possible to differentiate between the serologically distinguishable types AAV-1, AAV-2, AAV-3, AAV-4 and AAV-5. AAV viruses can either integrate into the genome of the host cell or replicate in the host cells in the presence of a helper virus. Adenoviruses were first of all found as possible helper viruses. In vertebrates, for example, adenoviruses form a group of more than 80 serologically distinguishable serotypes and contain an outer, icosahedral protein coat (capsid) and an inner, central DNA-protein body (core). The capsid is in turn composed of 252 subunits, so-called capsomers. In general, the adenoviruses have a diameter of approx. 70-90 nm and contain, as the genetic material a double-stranded, linear DNA at the 5xe2x80x2 and 3xe2x80x2 ends of which there are ITR regions (inverted terminal repeats). AAV replication (lyric phase) now requires, in particular, the expression of early adenoviral genes such as, e.g., the E1a, E1b, E2a and E4 genes and the VA RNA (Kotin, R. M. (1994) Human Gene Therapy, 5, 793-801). However, other helper viruses, such as the herpesviruses, which is a group of double-stranded DNA viruses which are pathogenic to humans and animals and which have a diameter of approx. 120-200 nm, with the capsid having an icosahedral structure and being composed of 162 capsomers, are also suitable. The herpesviruses can be divided into three subfamilies, with, for example, type I and type 2 herpes simplex viruses (HSV) belonging to the Alphaherpesvirinae, cytomegalovirus (CMV), for example, belonging to the Betaherpesvirinae, and Epstein Barr virus (EBV), for example, belonging to the Gamma-herpesvirinae.
In analogy with the retroviral vectors, one or more of the rep genes which are required for replication (e.g. rep 40, rep 52, rep 68 and/or rep 78) or the cap genes which are required for the capsid structure (e.g. VP-1, VP-2 and/or VP-3) can, for example, be replaced with the transgene(s) when preparing adeno-associated vectors. The ITR regions which are still present at the 5xe2x80x2 and 3xe2x80x2 ends are needed, as cis-active elements, for packaging the transgene into infectious, recombinant AAV particles and for the replication of the DNA of the recombinant AAV genome (Kotin, R. M. (1994), loc. sit.).
Cotransfection of a eucaryotic cell with two recombinant AAV plasmids and a helper virus (Chiorini, J. A. et al. (1995) Human Gene Therapy, 6, 1531-1541) is an advantageous method for preparing relatively large quantities of recombinant AAV particles. The first recombinant AAV vector contains the transgene(s) which is/are flanked by the two ITR regions. The second recombinant AAV plasmid contains the AAV genes which are required for preparing the particles (rep and cap genes). The absence of functional ITR regions in the second vector prevents the rep and cap genes being packaged into AAV particles and undesirable wild-type AAV thus being formed. Mammalian cells, for example COS-7 cells, which are permissive both for the recombinant AAV vectors and for the helper virus, for example adenovirus, i.e. which provide the prerequisites for infection and replication, are then transfected with the two recombinant AAV vectors and the helper virus. The adenovirus is particularly suitable for use as the helper virus since it can infect a broad spectrum of target cells and can replicate in the cells themselves. When the transfected cells are cultured, the AAV non-structural protein genes and the AAV structural protein genes are expressed, the transgene DNA is replicated and the recombinant AAV particles (rAAV particles) are packaged and assembled. The rAAV particles contain the transgene(s), which is/are flanked at both ends by the ITR regions, in the form of single-stranded DNA. At the same time, the helper virus replicates in these cells, something which generally ends, when adenoviruses are used as helper viruses, in the lysis and death of the infected cells after a few days. The resulting viruses (adenoviruses and rAAV particles) are either in part released into the cell culture supernatant or else remain in the lysed cells. For this reason, the cells are generally disrupted using cell disruption methods which are known to the skilled person, such as alternately freezing and thawing or by means of enzymic hydrolysis, for example with trypsin (Chiorini, J. A. et al. (1995), loc. sit.), in order to achieve essentially complete release of the viruses.
A significant disadvantage associated with preparing viral vectors using helper viruses is the formation of a mixed population of recombinant virus particles and helper viruses, which population has to be subjected to further purification. In particular, contaminations with adenovirus should be avoided or minimized when using viral vectors in gene therapy because of the potential pathogenicity.
The customary methods for depleting or eliminating adenoviruses, for example, in mixed populations containing AAV particles, for example, are density gradient centrifugation or heat inactivation, or a combination of the two methods. The mode of action of density gradient centrifugation is based, in this case, on a minor difference in the density of the adenoviruses (1.35 g/cm3) and the AAV particles (1.41 g/cm3) which can be exploited, for example, in CsCl density gradient centrifugation. Because of the small difference in the density of the viruses and the fact that the quantity of the adenoviruses is from approx. 10 to 100 times higher than that of the AAV particles, the regions in which the AAV particles and the adenoviruses are located after the CsCl density gradient centrifugation are very close to each other such that it is not possible to separate the two viruses quantitatively by carrying out one or more density gradients. Furthermore, the mixed population has to be present in a relatively small volume, since density gradient centrifugation can only be carried out using relatively low volumes. Routine use on an industrial scale is not therefore technically or financially feasible. In addition, it is not possible to separate the adenoviruses quantitatively from the mixed population.
The mode of action of heat inactivation is based on the adenoviruses and the AAV particles, for example, having different thermal stabilities. Heating a mixed population of adenoviruses and AAV particles to 56-65xc2x0 C. leads to more or less selective heat inactivation of the adenoviruses with there only being a slight loss in the activity of the AAV particles. However, the disadvantage of this method is that, when the preparation is used in gene therapy, the denatured adenovirus proteins which are still present are able to evoke a powerful cellular immune response in the patient (Smith, C. A. et al. (1996) J. Virol., 70, 6733-6740).
It is also known that filter membranes having a pore size which ensures that particles in the nanometer range are removed can be employed for separating off viral contaminants from pharmaceutical products which have to be virus-free (Scheiblauer, H. et al. (1996), Jahrestagung der Gesellschaft fur Virologie [Annual Conference of the Virology Society], Abstract No. P54). While Scheiblauer et al. (1996, loc. sit.) point out that the viruses are retained on the basis of their size, it was not reported that the different viruses were separated quantitatively.
The object of the present invention is therefore to provide a process in which different viruses present in a mixed population can be separated from each other and which can be used even in the case of relatively large volumes.
It has now been found, surprisingly, that a separation, which is essentially quantitative or is adequate for use in gene therapy, of viruses of different sizes is achieved, in a manner which is both simple and applicable on an industrial scale, by subjecting a virus-containing solution to one or more filtrations.
One part of the subject matter of the present invention is therefore a process for separating viruses of different sizes, with a virus-containing solution preferably being filtered through one or more filter membranes.
Advantageously, the filter membranes are composed of polyvinylidene fluoride, polytetrafluoromethylene, polypropylene, modified or unmodified polyether sulfone, cellulose acetate, cellulose nitrate, polyamide or regenerated cellulose, for example cuprammonium-regenerated cellulose, preferably of polyvinylidene fluoride. Examples of such membranes having a pore size which permits the removal of particles having a size of approx. 40-400 nm are the Ultipor membranes from Pall GmbH, 63303 Dreieich, which have pore sizes of approx 50 nm or approx. 100 nm, the Asahi Chemical""s Bemberg microporous membranes from Asahi Chemical Industry Ltd., Tokyo, Japan, which have pore sizes of approx. 15 nm, approx. 35 nm or approx. 72 nm, or corresponding membranes from Sartorius AG, 37075 Gottingen or Schleicher and Schuell GmbH, 37582 Dassel. In general, the filter membranes in accordance with the present invention have a pore size which permits the removal of particles of up to approx. 120 nm, in particular of up to approx. 60 nm, especially of up to approx. 50 nm, preferably from approx. 25 to 60 nm. The choice of a filter having a suitable pore size depends, first and foremost, on the sizes of the viruses to be separated, which viruses generally have a diameter of up to approx. 250 nm, preferably up to approx. 120 nm, in particular up to approx. 60 nm. An additional selection criterion when choosing a filter having a suitable pore size is the difference in the sizes of the viruses to be separated, which difference should in general amount to at least approx. 5 nm, preferably at least approx. 10 nm, in particular at least approx. 20 nm, especially at least approx. 30 nm and, most preferably, approx. 40 nm. Using the sizes of the viruses to be separated, and the difference in sizes resulting from this, as a basis, the skilled person can then select a filter which has a pore size which is suitable for separating the viruses which are to be separated. The efficacy of the selected filter(s) can then be readily tested routinely by means of simple filtration experiments.
Thus, a filter having a pore size which enables the titer of viruses having a diameter of more than approx. 50 nm to be reduced, preferably the Pall-Ultipor VF DV50 membrane or the Asahi-Planova 35 membrane, which have already been mentioned above, is suitable, in a particularly advantageous manner, for separating rAAV particles and adenoviruses. In particular, these filters make it possible not only to achieve an essentially quantitative removal of infectious adenoviruses from the mixed population but also to achieve a high yield, of approx. 70-90%, of rAAV particles.
Within the meaning of the present invention, the terms xe2x80x9cpore sizexe2x80x9d, xe2x80x9cremoval ratexe2x80x9d, xe2x80x9ceffective sizexe2x80x9d or xe2x80x9cpore size which permits particles having a particular minimum size to be removedxe2x80x9d, which are used by different filter manufacturers, are equivalent terms.
Within the meaning of the present invention, the term virus or viruses encompasses not only naturally occurring viruses or viruses which have been altered by genetic manipulation, i.e. so-called recombinant viruses, but also virus particles, i.e. both infectious and non-infectious viruses, virus-like particles (xe2x80x9cVLPxe2x80x9d), such as papillomavirus-like particles in accordance with WO 96/11272, and capsids which contain nucleic acids, but can also be empty, and parts thereof, in particular, one or more, preferably several subunits or capsomers, especially when several capsomers are associated or combined such that they constitute at least approx. 50%, preferably at least 80%, especially approx. 90%, of the capsid. The viruses have, in particular, an essentially spherical shape, in particular possessing icosahedral symmetry.
According to the present invention, the viruses can be classified, in accordance with their size, into the following three groups:
The first group represents viruses which have a diameter of up to approx. 60 nm and which are generally derived, for example, from Flaviviridae having a diameter of approx. 40-60 nm, such as, for example, flavivirus or hepatitis C virus (HCV), from papillomaviruses having a diameter of approx. 55 nm, such as human papillomaviruses 16 or 18 (HPV 16 and HPV 18), from papovaviruses having a diameter of approx. 45 nm, such as polyomavirus, from hepadnaviruses having a diameter of approx. 22-42 nm, such as hepatitis B virus (HBV), from picornaviruses having a diameter of approx. 22-30 nm, such as hepatitis A virus (HAV) or poliovirus, or from parvoviruses having a diameter of approx. 18-26 nm, such as adeno-associated virus (AAV) or rAAV particles having a diameter of approx. 25 nm.
The second group represents viruses which have a diameter of from approx. 60 nm up to approx. 120 nm and which are generally derived, for example, from influenza viruses having a diameter of approx. 80-120 nm, from retroviruses having a diameter of approx. 80-120 nm, such as onco-viruses, lentiviruses, such as HIV-1 or HIV-2, spuma-viruses or HTLV viruses, from adenoviruses having a diameter of approx. 65-90 nm, from reoviruses having a diameter of approx. 60-80 nm, such as coltivirus or rotavirus, or from togaviruses having a diameter of approx. 60-70 nm.
The third group represents viruses which have a diameter of from approx. 120 nm up to approx. 250 nm and which are generally derived, for example, from paramyxoviruses having a diameter of approx. 150-250 nm, or from herpesviruses having a diameter of approx. 120-200 nm, such as HSV-1 or HSV-2, CMV or EBV.
According to the present invention, the viruses of one of the abovementioned groups can particularly advantageously be separated from viruses of the other groups. However, different viruses which belong to one and the same group, out of the abovementioned groups, can also be separated from each other provided there is a sufficient difference in their sizes, preferably as already described in detail above. In particular, the viruses of the first group can be separated from the viruses of the second and/or the third group; especially, AAV particles can particularly advantageously be separated from adenoviruses and/or herpesviruses.
For example, the process according to the invention makes it possible, in a particularly advantageous manner, to remove adenoviruses, both in the case of material which has been prepurified, for example by means of one or more density gradients, and in the case of unpurified material, from the supernatants of an rAAV culture, for example, with the adenoviruses remaining in the retentate and the rAAV particles being present in the filtrate. Advantageously, a prefiltration, as described in more detail below, is carried out prior to the actual separation of the desired viruses.
Another preferred embodiment of the process according to the invention therefore extends to a prepurification of the virus-containing solution, for example by means of one or more density gradients and/or by means of one or more prefiltrations. Particular preference is given to a prepurification using one or more membrane filters which allow the viruses which are to be separated to pass but which nevertheless retain larger impurities. In general, the prepurification prevents, or renders more difficult, the clogging of the filter, which brings about the subsequent separation of the desired viruses, by constituents of the virus culture which are not removed, or are not adequately removed, even by centrifugation at low speed. In this connection, the membrane filter for prepurifying viruses of the abovementioned first group, for example, has a pore size of at least approx. 60 nm, while the membrane filter for prepurifying viruses of the first and/or second group has a pore size of at least approx. 120 nm and a membrane filter for prepurifying viruses of the first, second and/or third group has a pore size of at least approx. 250 nm. For example, a membrane filter having a pore size of approx. 100 nm, such as the Ultipor N66 filter (Pall GmbH, 63303 Dreieich), is particularly well suited for prepurifying a mixed population of rAAV particles and adenoviruses.
In another preferred embodiment, the pH of the virus-containing solution is adjusted with suitable buffers, for example tris.Cl buffers (tris(hydroxymethyl)amino-methane), to approx. 6-10, preferably to approx. 7.0-8.5, in particular to approx. 8.0, thereby making it possible, for example, to increase the yields, preferably the yields of AAV. It is also particularly preferred if the virus-containing solution additionally contains protein or polypeptide, for example in the form of fetal calf serum (FCS) or serum albumin, in connection with which the nature or origin of the protein is not important. In general, the proportion of the protein, for example in the form of FCS, is not more than approx. 30% (v/v), preferably approx. 15% (v/v), in particular approx. 10% (v/v).
In another preferred embodiment of the process according to the invention, at least approx. 1-10 ml, preferably approx. 2-5 ml, in particular approx. 3 ml, of the virus-containing solution is/are filtered per approx. 1 cm2 of filter surface, especially not more than approx. 2-2.5 ml of solution per approx. 1 cm2 of filter surface. In general, this achieves a high yield of rAAV particles, for example, while at the same time achieving the removal of the adenoviruses, for example. Particular preference is given to separating off cell constituents in the virus culture, prior to the actual separation, by means of centrifuging at low speed, for example at up to approx. 1000 times gravitational acceleration (1000 g), preferably at up to approx. 500 g, in particular at up to approx. 300 g. In connection with this, it is advantageous to subsequently carry out a prefiltration as already described in more detail above. In this way, it was possible, for example, after separating off the cell constituents at approx. 300 g and prefiltering the mixed population through a membrane filter having a pore size of approx. 100 nm, to determine the capacity of a filter having a pore size of approx. 50 nm to be 2 ml/cm2 of filter surface. Within the meaning of the present invention, capacity is understood as being the volume which, per unit area of filter surface, ensures a high is yield of the desired viruses.
The advantages of the process according to the invention are, in particular, a separation, which is simple and inexpensive and therefore also applicable on an industrial scale, of viruses of different sizes under particularly mild conditions, with the separation resulting, in a particularly advantageous manner, in a high yield of purified viruses. A further advantage of the present invention is that the viruses which have been purified in accordance with the invention can, for example, be employed directly as viral vectors for gene therapy since they are adequately free of other viruses, for example pathogenic viruses. Thus, it was not possible, for example, to detect any adenoviruses in an rAAV-containing solution, which had been purified in accordance with the process according to the invention, using a replication test in which the development of a cytopathic effect in permissive cells within a period of, for example, 14 days after coincubating with a corresponding solution would indicate the presence of infectious adenoviruses. Furthermore, use of the PCR reaction and DNA hybridization demonstrated that adenoviral nucleic acids were reduced by a factor of at least approx. 3xc3x97103, preferably approx. 103-104-fold. However, the small residual fraction of adenoviral DNA which remains in the filtrate is not associated with replication-competent adenoviruses and is consequently harmless. The decrease in adenoviral constituents, for example free adenovirus proteins and subviral particles, was particularly surprising since these constituents are perfectly capable of inducing a nonspecific immune response when the viral vectors are used in gene therapy in a patient and could consequently cast doubt on the therapy. In the case of adenoviruses, for example, the reduction in the titer, i.e. the factor by which the titer of the viruses to be removed was decreased, was, in accordance with the process according to the invention, more than 9 logs, i.e. after a mixed population of rAAV particles and 109 adenoviruses had been filtered in accordance with the invention, no adenovirus was then detected in the filtrate.
The following examples are intended to clarify the invention without limiting it thereto: